The present invention relates generally to methods and compositions for delivering foreign genetic material into cells. Specifically, it relates to a technique for receptor-mediated delivery of genes to cells. A gene delivery complex compatible with a specific type of targeted cell is formed from the foreign genetic material, a vector, and optionally, a carrier. The complex is then exposed to the cells under conditions permitting receptor-mediated endocytosis, resulting in the functional uptake, or transduction, of the foreign genetic material. The method is not only useful for in vitro, but also in vivo gene delivery to antigen presenting cells, specifically described as transcutaneous gene transfer to skin Langerhans cells. This technique is particularly useful for preventive and therapeutic genetic immunization when the foreign genetic material is an immunogen such as DNA encoding a substantial portion of the antigens and particles associated with an infectious virus, and where delivery by injection is undesirable.
The immune system for animals has two different but related responses, the cellular immune response and the humoral immune response. The cellular immune response produces T lymphocytes which kill cells having foreign identifying markers on their surface. Cells which have such identifying markers on their surface are said to xe2x80x9cpresentxe2x80x9d an antigen, and are referred to as antigen presenting cells (APCs). In addition, T lymphocytes also stimulate the humoral response by helping B cells, the precursors of plasma cells.
The humoral immune response results in the production by plasma cells of antibodies which act on specific molecules in solution. Antibodies (or immunoglobulins) are proteins synthesized by an animal in response to the presence of a foreign substance. They are secreted by plasma cells, which are derived from B lymphocytes (B cells). These soluble proteins are the recognition elements of the humoral immune response. Each antibody has specific affinity for the foreign substance that stimulated its synthesis. That is, the antibody has a segment or site, called an antigen binding site, which will adhere to the foreign substance. A foreign macromolecule capable of eliciting the formation of antibodies against itself is called an antigen. Proteins and polysaccharides are usually effective antigens. The specific affinity of an antibody is not for the entire macromolecular antigen, but for a particular site on it called the antigenic determinant or epitope. Antibodies recognize foreign molecules in solution and on membranes irrespective of the molecule""s context. The humoral immune response is most effective in combating bacteria and viruses in extracellular media. (The word humor is the Latin word for fluid or liquid.) One strategy for conferring immunity against disease is to expose the individual to one or more antigens associated with a virus or bacterium rather than use the actual virus or bacterium. Such a vaccine is known as a subunit vaccine, and it works particularly well to stimulate the production of antibodies.
T cells mediate the cellular immune response. In contrast to the humoral immune response, the cellular immune response destroys virus-infected cells, parasites, and cancer cells. The surface of T cells contain transmembrane proteins called T cell receptors that recognize foreign molecules on the surface of other cells. That is, T cells recognize antigen presenting cells (APCs). T cell receptors do not recognize isolated foreign molecules. The foreign unit must be located on the surface of a cell, and must be presented to the T cell by a particular membrane protein, one encoded by a highly variable chromosomal region of the host known as the major histocompatibility complex (MHC). The MHC encodes three classes of transmembrane proteins. MHC Class I proteins, which are expressed in nearly all types of cells, present foreign epitopes to cytotoxic T cells. MHC Class II proteins, which are expressed in immune system cells and phagocytes, present foreign epitopes to helper T cells. MHC Class III proteins are components of the process know as the complement cascade.
There are a variety of T cells, including cytoxic T lymphocytes (CTL, or killer T cells) which destroy cells which display a foreign epitope bound to an MHC protein. When the foreign-epitope-plus-MHC-protein binds to the T cell receptor, the T cell secretes granules containing perforin, which polymerizes to form transmembrane pores, thereby breaking the cell open, or inducing cell lysis. Other classes of T cells, called Helper T cells, secrete peptides and proteins called lymphokines. These hormone-like molecules direct the movements and activities of other cells. Some examples are Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interferons, Granulocyte-Macrophage colony-stimulating factor (GM-CSF), and Tumor necrosis Factor (TNF). The T cells are implicated in the complement cascade, a precisely regulated, complex series of events which results in the destruction of microorganisms and infected cells. More than fifteen soluble proteins co-operate to form multi-unit antigen-antibody complexes that precede the formation of large holes in the cells"" plasma membrane.
Expression of foreign genes in antigen presenting cells (APC) may be used to generate efficient CTL response in animals. Therefore, gene transfer and genetic modification of APC has potential to generate effective vaccine and therapeutic approaches against different diseases, including viral infections and cancer. Live recombinant virus vectors expressing various foreign antigens, such as pox viruses, adenoviruses, and retroviruses, can be used to elicit both humoral and cellular immune response by mimicking viral infection. Also, live attenuated (or, weakened) viruses have been proposed as vaccines. DNA vaccination strategy is also being explored. Different viral genes have been cloned into plasmid DNA and injected into muscles, skin, or subcutaneously. These constructs are able to express proteins and elicit both a cellular and humoral immune response.
It has been suggested that viral diseases may be responsive to the technique of genetic immunization. Certain cells, such as dendritic cells, are known to pick up antigens and migrate from the tissues of the body to the lymphoid tissues. There these cells present the antigens in the lymphoid organs: that is, they display a foreign epitope bound to an MHC protein. Such antigen-presenting cells (APCs) are a known part of the immune response mechanism. If cells such as a dendritic cells (DC) are modified so that they contain DNA encoding a virus which is infectious but incapable of efficient reproduction, they could not only present antigens in the classic sense, but also be manipulated to produce, or express, viral particles and a wide variety of viral proteins. A novel technology has been described in U.S. Ser. No. 08/803,484 xe2x80x9cMethods and Compositions for Protective and Therapeutic Genetic Immunizationxe2x80x9d which is incorporated herein by reference as if set forth in full. It discloses that genes of a replication-incompetent virus can be incorporated into antigen presenting cells which then migrate to the lymphoid organs and produce the full complement of viral antigens and viral particles, thereby triggering both humoral and cellular immune responses. It teaches that DC in the lymphoid organs may then express all viral antigens and produce xe2x80x9cauthentic lookingxe2x80x9d viral particles. These viral particles would therefore play a pivotal role in the generation of additional immune responses.
This reference describes in Example 13 xe2x80x9cin vivo transductionxe2x80x9d of cells including APC. In that example, several well known methods including viral and non-viral gene delivery are exemplified. In Example 14 xe2x80x9cin vivo transductionxe2x80x9d of cells including APC are described. These utilize (1) direct DNA injection; (2) injection of liposomes or virosomes containing the DNA; (3) direct intersplenic injection of Class 4 pox viruses; and (4) rectal and vaginal suppositories carrying gene delivery vehicles. However, this reference did not describe in detail the methods of in vitro and in vivo gene delivery. That is the subject of the present invention.
There is some evidence suggesting that genetic modification of APC will be effective to vaccinate both neonatal and adult animals and humans. Ridge et al. (Science 271: 1723-1726, 1996) have injected DC expressing a foreign antigen isolated from another animal intravenously into mice. Both neonatal and adult mice injected with these DC were able to generate good CTL killing of target cells. These experiments also demonstrated that DC expressing a foreign antigen can induce protective cell-mediated immune response which is able to eliminate infected cells in case of viral infections. In addition, these experiments demonstrated that DC-mediated immunization of neonates may be possible. These experiments did not use genetically modified cells, nor did they utilize several foreign antigens nor a virus as described in the present invention.
Sarzotti et al. (Science 271: 1726-128, 1996) demonstrated that low dose inoculation with viruses results in a protective immune response (Th1-type) which generates CTL response but high dose inoculation will result in a nonprotective (Th2-type) immune response which mainly generates antibodies. These CTL responses were very long lasting and also could be generated in neonates. High doses of virus might overwhelm and disarm T-cells before DC could activate the T-cells. Again, the route of administration, not through injection but through presentation by DC, is important. These findings are consistent with other results showing that exposure to low dose viruses provokes predominantly cellular (Th1-type) immune response. In macaques, a low dose SIV primed the Th1-type response without antibody production and protected animals against high dose challenge (Clerici et al. AIDS 8: 1391-1395, 1994). In humans, similar results were demonstrated by Rowland-Jones et al. (Nature Med 1: 59-64, 1995)
The process of modification of cells so that they contain foreign genetic material is called gene transfer, transfection or transduction. None of the papers cited herein have presented evidence of efficient gene transfer to antigen presenting cells, either in vitro or in vivo. As background for gene transfer into antigen presenting cells such as DC, several xe2x80x9clow efficientxe2x80x9d in vitro methods have been described, including liposome-mediated gene transfer; electroporation and retrovirus-vector- and adenovirus-vector-mediated gene transfers (Arthur, J. F et al. Cancer Gene Therapy. 4:1 17-21, 1997, Song, E. S. et al. Proc Natl Acad Sci U S A 94:5, 1943-8, 1997). All of these in vitro methods involve the isolation of large populations of cells which are treated in the laboratory with a gene delivery vehicle. All human or animal applications involve the reintroduction of these genetically modified cells. Therefore, in vitro gene delivery methods are not feasible for vaccination or treatment of large numbers of individuals. Known in vivo methods include intradermal or intramuscular injection of recombinant virus vectors and intradermal, subcutaneous and intramuscular injection of plasmid DNA. None of these methods have been shown to effectively deliver genes into antigen presenting cells, such as dendritic cells, much less delivery of genes through the skin into the Langerhans cells.